1. Field of the Invention
The present invention generally relates to agents that effect vasoprotection in mammals. More particularly, the invention pertains to anti-thrombotic agents that are adapted for localized rather than systemic administration. Still more particularly, the invention relates to recombinant adenovirus vectors containing a DNA sequence encoding a human tissue factor pathway inhibitor (TFPI) gene and to methods of making and using such vectors to effect local expression of TFPI in vascular smooth muscle cells at a specific blood vessel site.
2. Description of the Related Art
Pharmacological anticoagulation therapies are widely employed to deter thrombus formation in injured or atherosclerotic arteries. These therapeutic approaches typically employ physiological inhibitors of thrombin and require systemic administration of multiple drugs. However, the presence of anticoagulants in the circulating (systemic) blood is generally associated with increased bleeding risk. For instance, in clinical trials studying heparin and the thrombin inhibitor desirudin, most patients with acute coronary syndromes who developed intracranial bleeds had received aspirin, heparin or desirudin, and a thrombolytic agent (1-3). Those trials indicated that systemic blockade of multiple platelet/coagulation pathways is not without risk. Similarly, it is known that high doses of heparin are poorly tolerated in conjunction with potent platelet inhibition with c7E3 Fab (ReoPro) (4).
Short Term Local Administration of Antithrombotics
Since most conventional methods aimed at deterring thrombosis deposition act systemically and typically cause bleeding, some recent research efforts have focused on determining the feasibility of local anticoagulant treatment of predetermined "at risk" arterial sites, as opposed to treating the entire circulatory system. Local delivery of anticoagulant drugs has been attempted.
For example, the isolation of a portion of a vessel with a pair of angioplasty balloons and instillation of hirudin or heparin has been reported (4a). However, these methods are limited by uncertain drug delivery (given the systemic escape) and the short persistence of the antithrombotic drug in the vessel wall, given the diffusion gradient towards the vessel lumen. However, even in these localized treatments, no locally delivered antithrombotic drug has been reported to be present at the target site 48 hours after delivery. Points within the human circulatory system that are subject to injury, inflammation or atherosclerosis are especially likely targets for the local application of therapeutic anticoagulant agents, and include such specific sites as those subjected to angioplasty, stent or graft placement, or arteriovenous shunt.
For the purposes of this disclosure, "local" treatment, as distinguished from "systemic" treatment, means that a specific region, site or area within the blood circulatory system (especially a blood vessel) is the focus or target of the treatment and therefore receives the significant part of the treating agent, while the rest of the vessel and/or the circulatory system receive none or only an insignificant exposure to the treating agent. Short-term administration of antithrombins does little to passivate the injured artery, and allows thrombin generation to relentlessly proceed. In prior studies, for instance, it was found that short-term administration of the direct antithrombins failed to reduce restenosis rates after percutaneous coronary balloon angioplasty (5,6). This may be explained in part by experimental and clinical evidence suggesting that the thrombin inhibitors are not capable of inhibiting thrombin generation in the course of arterial thrombosis or in systemic procoagulant states (7,8). Furthermore, after withdrawal of short-term thrombin inhibitor therapy at 3-5 days, thrombin activity soon recurs (9,10).
Similar conclusions can be drawn from trials of short-term administration of synthetic inhibitors of GP IIb/IIIa integrin receptor (11). The administration of platelet IIb/IIIa integrin receptor blocker, c7E3 Fab (ReoPro), was effective in reducing early ischemic events in an early trial (12) and reduced the need for recurrent revascularization at 6-months. However, this was associated with increased bleeding at the time of the initial intervention (4). In a later trial, however, ReoPro failed to reduce the need for repeated revascularization at 6 months (13).
In summary, while effective during their administration, systemically given antithrombotic drugs are associated with increased hemorrhagic risk and require hospitalization associated with high cost, inconvenience, and additional risk of (hospital-acquired) infection; and, finally, do not passivate the thrombogenic lesion after the drug infusion is stopped (typically 3-5 days).
Anti-thrombotic Gene Therapy
Recent trials of systemic antithrombins to prevent restenosis after percutaneous revascularization suggest that there may be advantages to local antithrombotic gene therapy, which conventional drug therapy cannot presently match (5,6). Gene therapy potentially ensures the continuous in situ production of the foreign antithrombotic protein. Lee et al., in 1993, demonstrated that a replication-defective recombinant adenovirus can serve as an efficient vector for direct in vivo arterial gene transfer (14). Zoldhelyi et al. have previously described the adenovirus-mediated transfer of the cyclooxygenase gene (Ad.COX-1) as a localized anti-thrombotic agent (15). Cyclooxygenase is the rate-limiting enzyme in the synthesis of prostacyclin, an important vasoprotective molecule that inhibits platelet aggregation and vasoconstriction. Delivery of recombinant adenovirus to the artery at the doses used in the Ad.COX-1 study was associated with only minimal inflammation (15). Ad.COX-1 is a reasonable antithrombotic agent but has no direct influence on the thrombin-coagulation pathway involved in fibrin formation and smooth muscle cell proliferation contributing to restenosis after percutaneous balloon angioplasty. Also, platelet aggregation plays little role in venous thrombosis where thrombin inhibition is a highly effective approach (16).
Tissue Factor Pathway Inhibitor
Another approach to blockading platelet/coagulation pathways involves inhibiting thrombin activation via the tissue factor metabolic pathway. Tissue factor (TF), the cellular initiator of blood coagulation, is a transmembrane protein receptor exposed after vessel injury or after cytokine activation of endothelial cells and monocytes. Blood coagulation in the extrinsic pathway begins when the serine protease, activated factor VII (factor VIIa), which binds to its cofactor, TF, and the factor VIIa/TF enzyme complex activates by limited proteolysis of coagulation factors X and IX (17-19). On the membranes of activated platelets and endothelium, factor Xa then binds to factor Va, forming the prothrombinase complex, which in the presence of Ca.sup.2+ proteolytically converts prothrombin to thrombin (20). Factor IXa, also activated by the factor VIIa/TF complex, combines with factor VIIIa to activate in a second (intrinsic) pathway factor X. Thus, TF plays an initiating role for both the extrinsic and intrinsic pathway of thrombin generation (21).
Thrombin, the final product of the converging coagulation pathways, activates platelets and converts fibrinogen to fibrin, thereby stimulating formation of the fibrin-platelet clot. Thrombin not only activates platelets, converts fibrinogen to fibrin, and via factor XIII activation, stabilizes the fibrin clot, but also positively feeds back on its generation by activating platelets, factors V, VIII and XI (22-24). In addition, thrombin promotes release of P-selectin from storage granules of platelets and endothelial cells, contributing to platelet-leukocyte interaction and leukocyte rolling and migration into the vessel wall (25). Thrombin-activation of platelets promotes exposure of the platelet IIb/IIIb integrin receptor (26). Activation of this receptor mediates platelet-platelet and platelet-vessel wall interactions and, by recruiting additional platelets, thrombin allows for its generation to be further amplified.
Tissue factor pathway inhibitor (TFPI) inhibits thrombin generation at several steps. TFPI inhibits factor VIIa/TF, thereby initiating thrombin generation after vascular injury or cell activation by cytokines, and also inhibits the factor Xa/prothrombinase complex, thereby interrupting thrombin generation at its major amplification step (27,28).
The mature TFPI is composed of 276 amino acids and contains three tandem Kunitz-type protease inhibitory domains, in addition to an acidic amino-terminal and a basic carboxy-terminal region (27-29). End-linked glycosylation occurs at one or more of three potential sites, but the role of this and other post-translational modifications is not yet known (27,30).
Initially, the second Kunitz-type domain interacts with factor Xa, by binding with 1:1 stoichiometry at or near the factor Xa active serine site. This TFPI/factor Xa interaction, then, promotes efficient binding of the first Kunitz-type domain to the factor VIIa/TF complex, resulting in a quaternary factor Xa/TFPI/factor VIIa/TF complex (27,28,31 ). The basic carboxy-terminal region of TFPI also contributes to high-affinity binding of factor Xa (32,33). While the inhibition of factor VIIa/TF by physiological concentrations of TFPI is dependent on initial binding to factor Xa, TFPI at 50-fold greater concentration can inhibit factor VIIa/TF in the absence of factor Xa. The function of the third Kunitz-type domain of TFPI is not clear and its deletion has no significant effect on factor Xa or factor VIIa/TF inhibition (27,28).
The concentration of TFPI in the plasma is about 2 nM, or 68-82 ng/mL (the apparent molecular weight of TFPI in plasma ranging from 34-41 kD) (27,28). Over 90% of circulating TFPI is bound to the lipoproteins, LDL, HDL, and Lp(a). Platelets contain about 10% of TFPI in 1-5 the circulating blood and release TFPI upon aggregation initiated by thrombin and other agonists (34). Plasma TFPI concentrations increase 2 to 4-fold after infusion of heparin, presumably by displacement of TFPI from heparin sulfate or other glycosaminoglycans on the surface of endothelial cells (35,36). TFPI associated with the endothelium may be important in local regulation of coagulation and its release by heparin may contribute to systemic anticoagulation (37). Conversely, heparin (in the presence of calcium) enhances the inhibition of factor Xa by full-length and carboxy-terminus truncated TFPI (28).
The tissue distribution of TFPI has not been fully elucidated (38). By immunohistochemistry, TFPI was detected in one study in megakaryocytes and microvascular endothelium but not in the endothelium of large and medium-sized vessels (39). Others recently reported the presence of endothelial TFPI in normal and atherosclerotic arteries but detected no TFPI in the smooth muscle cells of the medial layer (40). The inventors were also unable to detect TFPI in the conditioned medium of cultured human VSMC (See discussion in "Examples" infra.)
Mice with inactivated TFPI gene are subject to intrauterine lethality as a result of bleeding due to disseminated intravascular coagulation (41). Therefore TFPI likely plays an indispensable role among the endogenous antithrombotic molecules. However, physiological concentrations of TFPI do not completely inhibit thrombin generation. After prothrombotic stimuli (such as gram-negative sepsis) activate the factor VIIa/TF complex, disseminated intravascular coagulation can occur, consistent with the ability of physiological TFPI levels to inhibit factor VIIa/TF-initiated thrombin generation only after some factor X activation and prothrombinase complex formation have occurred (28). In contrast, pharmacological concentrations of TFPI inhibit the TF/factor VIIa complex in Xa-independent fashion and were reported to prevent disseminated intravascular coagulation after gram-negative bacterial sepsis (42,43) and thrombus formation after vascular injury (44-46). Experimentally, systemic administration of TFPI accelerates pharmacological lysis of arterial thrombi (47,48) and attenuated neointima formation after percutaneous balloon angioplasty (49,50).
Expression of tissue factor is tightly regulated (17,18) because TF is the common membrane receptor involved in blood vessel formation (62), hemostasis, and thrombosis at sites of vessel injury, inflammation and atherosclerosis. In healthy vessels, TF is mainly present in the adventitial layer (51). In contrast, in atherosclerosis, TF is expressed in the medial smooth muscle cell layer and the intimal plaque as well (52). TF is also upregulated acutely by shear stress (53), oxygen-free radicals generated during post-ischemic reperfusion (54), balloon injury (55) and, in vitro, by lipopolysaccharide, phorbol ester, interleukin-1, tumor necrosis factor, and other cytokines (17,18,56-61). The increased TF burden in the atherosclerotic vessel wall not only heightens the thrombotic risk, but also contributes to other problems. Factor Xa and thrombin, activated in TF-initiated pathways, are potent mitogens for vascular smooth muscle cells (63-65) and thereby may promote formation of the fibrous cap and restenosis after arterial revascularization interventions. TF may also directly contribute to neointimal smooth muscle cell accumulation through chemotactic effects (66). Thrombin activates matrix metalloproteinases involved in intimal smooth muscle cell accumulation and plaque rupture (67,68) and, through P-selectin release (25) may promote accumulation of inflammatory cells contributing more TF to the atherosclerotic plaque. Thus, TF initiates pathways, which lead to atherosclerotic plaque instability, rupture, thrombosis and exuberant proliferative "repair" and may promote thrombosis, inflammation, and intimal proliferation after percutaneous revascularization interventions as well.
None of the known anti-thrombotic methods employ gene therapy to achieve local expression of an anti-thrombotic agent specifically targeting the tissue factor pathway of thrombin generation. What is needed is an alternative or superior anti-thrombotic agent that can provide its therapeutic effects without incurring hemorrhagic risk. It is also desirable to have such an agent that can provide vessel site-specific anti-thrombotic activity and deter or prevent restenosis after balloon-injury. A method employing such an agent should be able to provide long-term therapeutic effects without increasing hemorrhagic risk, and without the need for co-administering multiple drugs.